Browsing by Subject "Antiviral Agents"
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Item Characterization of the Antiviral Effector IFI6(2018-11-26) Richardson, Ryan Blake; Yan, Nan; Schoggins, John W.; Levine, Beth; Pfeiffer, Julie K.The innate immune response is a critical line of host defense against invading pathogens. The production of interferon (IFN) and the subsequent expression of interferon stimulated genes (ISGs) are major contributors to the innate immune response, which establish an antiviral state in the cell. Flaviviruses such as dengue virus, Zika virus, and West Nile virus rely intimately on host pathways for completing a replication cycle, and have developed strategies to overcome the inhibitory effect of the innate immune response. To identify host factors required during an IFN response to flavivirus infection, a genome-wide CRISPR screen was carried out. Two of the top hits from the screen were IFI6, a previously identified ISG long predicted to be antiviral, and BiP, a luminal chaperone in the endoplasmic reticulum (ER). I questioned whether IFI6 was important for the antiviral response to flaviviruses and sought to investigate its role during infection. I confirmed the results from the CRISPR screen and showed that cells lacking IFI6 were insensitive to IFN, suggesting a key role in the innate immune response to flaviviruses. This was complemented by overexpression studies which showed IFI6 is potently inhibitory to flavivirus infection. I further demonstrated that BiP is required for an intact IFN response and importantly mediates expression of IFI6, which it binds in a chaperone-dependent manner. I also showed that IFI6 is localized to the ER and is an integral membrane protein. Importantly, IFI6 acts during the flavivirus life cycle to inhibit replication and formation of replication complexes, which are formed by rearrangement of ER membranes. IFI6 specifically inhibits flaviviruses, since other viruses that replicate at the ER such as hepatitis C virus (HCV) are not affected by IFI6. I hypothesize the key to this specificity lies in the orientation of the replication complexes - HCV complexes extend outwards into the cytoplasm while flaviviruses bud inwards into the lumen. Taken together, these data support a model where IFI6 is sensitive to membrane alterations specifically induced by flaviviruses but not other viruses, which provides the innate immune response with a potent and specific ISG to block viral infection.Item Chemical Inhibition of RNA Viruses Reveals REDD1 as a Host Defense Factor(2013-08-07) Mata, Miguel Angel; Roth, Michael G.; White, Michael A.; Lum, Lawrence; Fontoura, BeatrizInfluenza (flu) is a contagious infectious respiratory illness. The flu can cause from mild to life-threatening illness. The current therapeutic intervention strategies to prevent or treat influenza infection are not sufficient in the event that a pathogenic virus strains reaches pandemic proportions. Therefore, the development of anti-influenza therapeutic modalities is critical to respond to a future influenza pandemic. In this study, a chemical genetics approach was taken to identify inhibitors of NS1, a major influenza A virus virulence factor that inhibits host gene expression. A high-throughput screen of 200,000 synthetic compounds identified small molecules that reversed NS1-mediated inhibition of host gene expression. A counterscreen for suppression of influenza virus cytotoxicity identified naphthalimides that inhibited replication of influenza virus and vesicular stomatitis virus (VSV). The mechanism of action occurs through activation of REDD1 expression and concomitant inhibition of mammalian target of rapamycin complex 1 (mTORC1) via TSC1–TSC2 complex. The antiviral activity of naphthalimides was abolished in REDD1−/− cells. Inhibition of REDD1 expression by viruses resulted in activation of the mTORC1 pathway. REDD1-/- cells prematurely upregulated viral proteins via mTORC1 activation and were permissive to virus replication. In contrast, cells conditionally expressing high concentrations of REDD1 downregulated the amount of viral protein. Whole animal studies revealed REDD1-/- mice are highly susceptible to virus infection. Influenza infection of REDD1-/- mice results in decreased TLR7 and MHC class II expression by dendritic cells and macrophages. In addition, excessive inflammatory cell infiltration in the lungs of REDD1-/- infected mice was observed. Preliminary evidence suggests a potential defect in NF-κB signaling upon influenza virus infection in REDD1 deficient mice. Thus, REDD1 is a new host defense factor, and chemical activation of REDD1 expression represents a potent antiviral intervention strategy. Our studies also reveal passage immortalization of REDD1-/- MEFs require loss of the type I IFN response pathway as these cells are unable to induce the expression of interferon genes and interferon inducible genes when challenged with synthetic dsRNA. In contrast, primary or SV40 large T antigen transformed REDD1-/- MEFs activate a type I IFN response when exposed to synthetic dsRNA.Item Decreased microRNA-122 Levels with HCV Clearance in HIV-HCV Co-Infections(2013-01-22) Dubin, Perry H.; Yuan, Hejun; Devine, Robert K.; Jain, Mamta K.; Hynan, Kinda S.; Lee, William M.BACKGROUND AND AIMS: Micro RNA-122 (miR-122) is under investigation as a target for direct antiviral agents against the hepatitis C virus (HCV), and as a biomarker for both cancer and acute liver injury. Previous data suggest HCV mono-infection is associated with increased serum miR-122 levels. This study sought to determine outcomes in regard to miR-122 levels following clearance of HCV in human immunodeficiency virus (HIV) co-infected patients. METHODS: Nine HCV-HIV co-infected patients undergoing antiviral therapy were treated with interferon and ribavirin for 48 weeks between January 2009 and March 2011, and had serial miR-122 levels measured in triplicate from serum with mirVanaTM PARISTM kit according to the instructions from the manufacturer (Ambion, AM1556). Values were measured at baseline, 1 week, 4 weeks, end of treatment (EOT; 48 weeks), and at 24 weeks after treatment completion (SVR24). SAS V9.3 was used to analyze these data. Change from baseline (copies/μL) was calculated as Log10 (Baseline)-Log 10(time), where time was 1 week, 4weeks, EOT, and SVR24; a repeated measures ANOVA was used to compare the results over time for the patients. If the ANOVA was found significant, post hoc, pairwise comparisons were used to examine change from baseline across the four time points. RESULTS: Six of nine achieved SVR24, 1 was undetectable at EOT but relapsed, and 2 patients were non-responders. Among the 6 patients achieving SVR, all showed a decrease in miR-122 levels between 0.16 and 1.46 logs, between baseline and SVR24. The ANOVA confirmed a significant decrease in miR-122 levels from 1 week to SVR24 (p=0.0225). Significant pairwise comparisons for change from baseline were found at 1 week versus SVR24 (p=0.0063), 4 weeks versus SVR24 (p=0.0086), and EOT versus SVR24 (p=0.0458). CONCLUSION: Clearance of chronic HCV is associated with decreased miR-122 levels in HIV co-infected patients and was not improved in patients with continued infection who failed to respond to treatment.Item Discovery of Unique Antiviral Pathways in a Bat Viral Reservoir, the Black Flying Fox(2018-04-03) De La Cruz Rivera, Pamela Cristina; Alto, Neal; Schoggins, John W.; Pfeiffer, Julie K.; Russell, David W.Bats are asymptomatic reservoirs for a number of viral pathogens. How they manage to host highly pathogenic viruses such a Nipah and Marburg without showing clinical symptoms remains unclear. One of the earliest defenses vertebrates use to control viral infections is the interferon response. Upon viral infection, cells produce interferon which inhibits viral infection through downstream effectors called interferon stimulated genes. I questioned whether bat interferon stimulated genes contained unique properties that would make bats less susceptible to damage from viral infection. To address this, I used genetic tools to identify which interferon stimulated genes were expressed in cells from the black flying fox (Pteropus alecto). I found that RNASEL is uniquely induced in cells from the black flying fox, and that its activation is important for preventing viral infection. To determine if any bat interferon stimulated genes had evolved especially potent antiviral properties, I compared a group of bat and human interferon stimulated genes in a high-throughput format and discovered that bat IRF7 is more antiviral than human IRF7. Further studies demonstrated that bat IRF7 is active even in uninfected cells, and can induce a subset of protective antiviral genes without signaling through interferon. This function was in part due to unique serine residues at the C-terminal regulatory region of the protein that confer constitutive activity to bat IRF7. This work has uncovered two different mechanisms by which antiviral responses between bat and human hosts differ, and provides insight regarding how bats manage to keep numerous viral infections under control.Item Herpes simplex infections: targets for new antiviral strategies(1982-04-22) Murphy, F. KevinItem Host-Based Mechanisms of Ribavirin Resistance: Implications In Treatment Response of Hepatatis C Virus Infection(2011-02-01) Ibarra, Kristie Dawn; Pfeiffer, Julie K.Many individuals infected with hepatitis C virus (HCV) fail to respond to therapy, resulting in the development of chronic infection and increased risk for fibrosis, cirrhosis, and hepatocellular carcinoma. The current standard of care consists of pegylated interferon and ribavirin (RBV), a nucleoside analog. While RBV improves treatment outcome, and will likely be an important component of therapy with next-generation viral inhibitors, RBV’s mechanism is controversial. Most of RBV’s proposed mechanisms require RBV import into cells. Therefore, we examined whether host-based RBV resistance develops through reduced cellular uptake, analogous to chemotherapy resistance in some cancers. We examined the effect of host-based RBV resistance on a model RNA virus, poliovirus, HCV replication in cultured hepatoma liver cells, and whether RBV resistance develops in HCV patients. When liver cells permissive for poliovirus or HCV replication were exposed to RBV, resistance developed through reduced activity of the ENT1 nucleoside transporter, and antiviral efficacy was reduced. Importantly, RBV uptake significantly declined in HCV peripheral blood mononuclear cells (PBMCs) following four weeks of therapy. Furthermore, maintenance of RBV uptake correlated with rapid treatment response. Our results uncovered a novel form of antiviral drug resistance, suggesting that host-based RBV resistance develops in HCV patients undergoing therapy and that maintenance of RBV uptake may contribute to rapid viral clearance.Item Identification of Receptor Transporting Proteins As Conserved Antiviral Effectors in Vertebrates(2021-05-27) Boys, Ian Nicholas; Shiloh, Michael; Alto, Neal; Yan, Nan; Hancks, Dustin C.; Schoggins, John W.Viruses and their hosts are engaged in "genetic arms races" in which each side attempts to gain the advantage over evolutionary time. Results of these conflicts are wide-ranging: viruses diversify, hosts establish species-specific barriers to some viruses while remaining susceptible to others, and the lines for future genetic conflicts are drawn. In mammals, many antiviral effectors -- proteins that directly inhibit viral infection -- show species- or lineage-specific properties which are believed to be the result of past or ongoing conflicts. Bats harbor a greater diversity of viruses than any other mammalian order, and a growing body of research has described unique adaptations in bats that are in part responsible for, and perhaps a response to, this unique status. We hypothesized that the frequent encounters between bats and viruses would drive unique adaptations in the antiviral effectors that serve on the front lines of virus-host genetic conflicts. We identified RTP4 from the bat Pteropus alecto as a potent inhibitor of flavivirus infection. Mechanistic studies determined that RTP4 is an RNA-binding protein that associates with flavivirus replication machinery, binds replicating viral RNA, and suppresses viral genome amplification. Phylogenomic analysis revealed that RTP4 has evolved under positive selection in several mammalian lineages, consistent with a model in which host-virus conflicts have shaped its evolution as a restriction factor not only in bats but across mammals. We assessed the antiviral efficacy of diverse mammalian RTP4 orthologs and found that orthologs exhibit striking patterns of antiviral specificity. Further highlighting the specificity of the host-virus arms race, experimental evolution demonstrated that a flavivirus can mutate to escape RTP4-imposed restriction in a species-specific manner. In follow-up work, we identified signatures of positive selection in several non-mammalian RTP homologs, indicative of a putative role in innate immunity. We screened a collection of vertebrate RTPs against a panel of viruses and identified antiviral RTPs in the African clawed frog, Xenopus laevis. These antiviral Xenopus RTPs exhibit mosaic phenotypes that resemble those of mammalian RTP4 orthologs. Within the context of our findings with mammalian RTP4, these data suggest that Receptor Transporter Proteins are involved in host-virus genetic conflicts outside of Mammalia.Item Insights into Virus-Host Interactions: Regulation of Cytokine Signaling Is a Virulence Determinant of West Nile Virus(2007-08-08) Keller, Brian Christopher; Gale, Michael, Jr.West Nile virus (WNV) has rapidly become a pathogen of global importance over the past two decades. Its recent association with severe neurological disease and emergence in the Western Hemisphere suggest that the virus has acquired the ability to effectively evade host immune defenses. One of the earliest steps in controlling viral infection occurs through the action of interferon (IFN) and its downstream activation of an antiviral state within the infected cell and neighboring tissue. To begin to understand how WNV evades host defenses, studies were initiated to examine the interaction of virulent (TX02) and avirulent (MAD78) WNV strains with the host IFN system. Compared to TX02, MAD78 replicated at lower levels in cultured human cells, was highly sensitive to the antiviral actions of IFN in vitro and demonstrated a completely avirulent phenotype in wild-type mice. In contrast to TX02 and other pathogenic forms of WNV, MAD78 was defective in its ability to disrupt IFN-induced JAK-STAT signaling. However, replication of MAD78 was rescued in cells lacking a functional IFNalpha /beta receptor (IFNAR). Consistent with this, MAD78 virulence was unmasked upon infection of mice lacking IFNAR. The regulation of IFN signaling was multifactorial involving a combination of viral and host factors. In particular, overexpression of various proteins from the pathogenic TX02 strain and the nonpathogenic MAD78 strain attenuated signaling to an IFN-responsive promoter, suggesting that viral products from both strains are capable of contributing to the IFN signal block. Differences between TX02 and MAD78 were identified, however, when host suppressors of cytokine signaling (SOCS) proteins were shown to be differentially upregulated by the two strains. Furthermore, expression of a dominant-negative form of SOCS1 partially restored IFN signaling during TX02 infection indicating that SOCS proteins do in fact participate in virus-induced signaling suppression. Importantly, WNV regulation of signaling was not restricted to IFNalpha /beta but included IFNgamma and IL-6, cytokines that similarly utilize JAK-STAT. These studies demonstrate novel insights into the complex interactions that occur between a virus and its infected host cell and may allow for the identification of viral and cellular targets for the development of improved therapeutics and new vaccine strategies.Item [UT Southwestern Medical Center News](2011-09-26) Bolles, Debbie